Project Summary Reversible phosphorylation of proteins on tyrosine controls the execution and regulation of many cellular processes. A proper level of tyrosyl phosphorylation is critical for these processes and is controlled by the opposing actions of protein tyrosine kinases and protein tyrosine phosphatases (PTPs). A large number of PTPs (>100) have been identified and their importance in physiological and pathological processes has been demonstrated unambiguously. However, their mechanisms of action in these processes remain unclear. To understand their mechanisms of action, a critical first step is to identify their protein substrates involved in these processes, a currently very challenge task. This project describes the development and application of a chemical/bioinformatics approach to the identification of PTP substrates. In this approach, the substrate specificity of PTP is systematically determined by screening a combinatorial peptide library and the consensus motif(s) is used to search protein and genomic databases to identify potential protein substrates. The candidate proteins are subsequently validated as genuine PTP substrates (or rejected as false positives) by conventional cellular assays. In this project, we will focus our studies on three classical, non-receptor PTPs: the prototypical PTP1B and two Src homology 2 (SH2) domain-containing PTPs, SHP-1 and SHP-2. It consists of three specific aims. Specific Aim 1 is to further develop the combinatorial peptide library method and determine the substrate specificity of PTP1B, SHP-1, and SHP-2. Specific Aim 2 is to identify the in vivo protein substrates of PTP1B, SHP-1, and SHP-2. Specific Aim 3 is to characterize SHP-2 mutants that are involved in human diseases. The binding and substrate specificity of the SH2 and PTP domains in SHP-2 mutants will be determined and the resulting information will be utilized to identify any protein substrates abnormally acted upon by SHP-2 mutants.